Our objective is to determine the molecular basis for impaired insulin signaling in a group of genetic syndromes characterized by severe insulin resistance. Experiments will focus on cultured skin fibroblasts from at least 12 such individuals who exhibit a range of clinical phenotypes. Biochemical, immunologic and molecular genetic probes will be used to define specific lesions of insulin and IGF receptors, glucose transporters, and other signaling components amenable to study. Specific aims include the following: 1) To define the consequences of the signaling defect in each mutant cell line by measuring insulins capacity to act on a range of distinct pathways; 2) To determine the functional state of the insulin receptor kinase, measuring insulin stimulated receptor autophosphorylation, ability of the receptor to phosphorylate a synthetic kinase substrate, as well as in vivo phosphorylation of a 185,000 MW protein that is a possible endogenous substrate of the receptor kinase. Through comparisons of defects in bioactivity and kinase activity, it may be possible to assign specific functional meaning to specific phosphorylation events; 3) To use IGF-I and monoclonal antibodies to the IGF-I receptor to determine the state of signaling through the Type I IGF receptor in these cells. We will determine the degree to which insulin stimulates through this receptor in these insulin resistant cells, and whether IGF receptor signaling is impaired in some of these patients who display abnormal growth; 4) To use the cloned human insulin receptor cDNA to define the genetic basis for insulin receptor dysfunction in these cells. This will include measurement of steady state receptor mRNA levels, and application of RNAase protection assay and restriction fragment length polymorphism analysis to detect mutations in the receptor gene. In addition, we will clone and sequence the cDNA's of the most interesting putatively abnormal receptors. These cloned cDNA's will be used to further define the functional receptor defects through transfection into receptor deficient cells; 5) To use a cDNA probe for the glucose transporter and antibodies to this protein to investigate possible defects in the structure or function of the transporter in insulin resistant cell lines. This is of special interest in one line in which we have defined an isolated defect in hormone stimulated glucose transport, the first instance of such a natural mutation to be described.